Although there has been considerable progress in lung transplant biology, post-transplant ischemia-reperfusion (IR) injury remains the major source of early mortality. Although circulating leukocytes are known to be important in lung IR injury, the role of resident lung leukocytes remains unknown. Thus our long-term goal is to understand the mechanisms of cytokine- and leukocyte-mediated acute lung IR injury. This Project focuses on the role of resident, interstitial lung leukocytes and cytokine mediators in the early phase of lung IR injury by using an in situ, buffer-perfused mouse lung IR model. This project will test the overall hypothesis that it is primarily the resident pulmonary macrophages which are activated by IR, produce TNF-alpha, and set the stage for initiation of full-blown lung IR injury. In addition, inhibition of macrophage activation by ATL- 303 activation of adenosine A2A receptors should ameliorate the majority of acute lung IR injury. Specific Aim 1 will identify the resident lung leukocytes (macrophages, neutrophils, lymphocytes) that are critical for induction of the early, acute phase of lung IR injury. We hypothesize that acute lung IR injury is primarily initiated by the pulmonary macrophages. Specific Aim 2 will determine key early transcriptional events (NF-kappaB activation) and cytokine/ chemokine activation (TNF-alpha, IFN-gamma, IL-1, MIP-1alpha, MCP-1, and RANTES) leading to acute lung IR injury, and identify the responsible leukocytes. We hypothesize that injury is largely, but not solely, initiated by macrophage-produced TNF-alpha and TNF-alpha-mediated signaling via cytokines, chemokines, and transcription factors. Specific Aim 3 will determine if activation of adenosine A2A receptors on resident leukocytes ameliorates acute lung IR injury independent of circulating leukocytes. We hypothesize that A2A receptor activation primarily on pulmonary macrophages will ameliorate lung IR injury independent of circulating leukocytes. Even in the most experienced hospitals, IR injury continues to be a major cause of morbidity and mortality early after lung transplantation. The proposed studies will directly address the causes IR injury with the hopes of initiating further studies focusing on therapeutic strategies aimed at preventing or ameliorating acute lung IR injury.